OBJECTIVE: To investigate effects of antioxidant stress protein heme oxygenase-1 (HO-1) on lipopolysaccharide (LPS)-induced endoplasmic reticulum stress (ERS) of rat hepatocytes. METHODS: The BRL cells (rat hepatocyte cell line) were cultured. The hepatocytes were treated with LPS, LPS+HO-1 siRNA, HO-1 siRNA and PBS solution, respectively. The cell viability was measured by trypan blue exclusion test. The apoptosis cells were detected by the fluorescent dye Hoechst 33258. Expressions of GRP78, CHOP, caspase-12 and HO-1 were detected by Western blotting. RESULTS: LPS caused an increase of HO-1 protein expression of rat hepatocytes in a dose-dependent and time-dependent manner, a up-regulation of GRP78, CHOP and caspase-12, a decrease in cell viability, and an increase in apoptosis rate of hepatocytes. Pretreatment of HO-1 siRNA inhibited the up-regulation of LPS-induced HO-1, however, aggravated ERS and cellular injury. CONCLUSION HO-1 inhibites ERS-mediated cellular injury of rat hepatocytes induced by LPS.
Animals ; Apoptosis/physiology* ; Endoplasmic Reticulum/metabolism* ; Endoplasmic Reticulum Stress/physiology* ; Heme Oxygenase-1/pharmacology* ; Hepatocytes/metabolism* ; Lipopolysaccharides/pharmacology* ; Rats

OBJECTIVETo investigate the effects of peroxisome proliferator activated receptor-alpha (PPAR-a) activation on oleic acid (OA)-induced steatosis and hepatic expression of heme oxygenase-1 (HO-1) using an in vitro cell model system.

METHODSA steatosis human hepatocyte in vitro model system was established by treating HepG2 cells with 0.2 mmol/L of oleic acid for 24 hours. The steatosis cells were then divided into four groups for an additional 24 hours of treatment with 0.2 mmol/L of oleic acid alone (model control group) or with 5, 10 or 50 pnol/L of fenofibrate (FF, a selective PPAR-a agonist; experimental groups). Untreated HepG2 cells served as non-steatosis controls. Effect of PPAR-a activation on fat accumulation was detected by Oil Red O staining and on intracellular triglyceride (TG) levels by enzymatic assay. mRNA and protein expression of PPAR-alpha and HO-1 were quantified by real-time PCR and immunocytochemistry, respectively. One-way ANOVA and the LSD t-test were used for between-group comparisons, and correlation analysis was performed with the Pearson's correlation coefficient.

RESULTSThe steatosis model control cells showed significantly increased TG deposition (379.98 +/- 23.19 mg/g protein, vs. non-steatosis controls F = 148.56, P< 0.01), significantly decreased mRNA and protein expression of PPAR-alpha (0.42 +/- 0.38,F= 177.64,P< 0.01 and 0.47 +/- 0.14, F= 120.76,P< 0.01) and HO-1 (0.36 +/- 0.66, F= 74.77,P< 0.01 and 0.26 +/- 0.10,F= 119.90,P<0.01). FF (5, 10 and 50 micromol/L) inhibited the steatosis induced by OA in a concentration-dependent manner (294.00 +/- 19.80, 250.33 +/- 9.96, and 196.99 +/- 9.14, F = 148.56, P <0.01) and increased the mRNA and protein expression of PPAR-alpha (0.55 +/- 0.65, 0.85 +/- 0.61, and 1.31 +/- 0.36,F= 177.64,P< 0.01; 0.82 + 0.11, 1.31 +/- 0.16, and 1.75 +/- 0.13, F= 120.76,P <0.01) and HO-1 (0.62 +/- 0.05, 0.84 +/- 0.07, and 1.30 +/- 0.11,F= 74.77,P <0.01; 0.44 +/- 0.08, 0.81 +/- 0.08, 1.20 +/- 0.10,F= 119.90,P< 0.01).

CONCLUSIONActivation of PPAR-a prevents OA-induced steatosis in HepG2 cells, and HO-1 may function as a downstream effector of this mechanism.

Fatty Liver ; chemically induced ; Heme Oxygenase-1 ; metabolism ; Hep G2 Cells ; Humans ; Oleic Acid ; pharmacology ; PPAR alpha ; metabolism ; Triglycerides ; metabolism

The aim of this research is to investigate the influence of microencapsulation on the expression of the oxidative stress genes and exogenous regulation of HepG2 cells. We compared the expression of hemeoxygenase-1 (HO-1) and glutathione S-transferases-A1 (GST-A1) in HepG2 cells under different culture conditions through real-time PCR. The effects of exogenous antioxidants on cell viability and albumin levels were also evaluated through MTT assay and ELISA assay. The results showed that after culturing for 6 and 16 days, the expression levels of HO-1 in encapsulated cells were approximately 4.9 and 3.1 times higher than that of monolayer cells at the same culture period; As for the expression levels of GST-A1, they were elevated to 11.2 and 33 times of monolayer cells (P < 0.05). Accordingly, we found that NAC at 5-10 mmol/L significantly increased the viability by 40%-70% and the biosynthetic function by 20%-30% in microencapsulated HepG2 cells (P < 0.05). GSH increased the viability of the encapsulated cells by 20%-55% and the biosynthetic function by 15% (P < 0.05). In conclusion, oxidative stress exists in the microcapsules and affects genes expression. Exogenous antioxidants can prevent the inhibition effects of oxidative stress on cellular growth.
Antioxidants ; pharmacology ; Cell Survival ; Gene Expression Regulation ; Glutathione Transferase ; metabolism ; Heme Oxygenase-1 ; metabolism ; Hep G2 Cells ; Humans ; Oxidative Stress

AIMTo explore the effect of ligustrazine injection on the expression of heme oxygenase-1 (HO-1) in rabbits with pulmonary ischemia/reperfusion injury after.

METHODSSingle lung ischemia/reperfusion injury animal model was used in vivo. Twenty rabbits were randomly divided into two groups( n = 10, in each), pulmonary ischemia/reperfusion injury (I/R) group and I/R + ligustrazine injection (LGT) group. The tissue slides were stained by immunohistochemistry (IHC) and in situ hybridization (ISH) for HO-1 to detect the expression of HO-1 in lung and to analyze the absorbance, wet to dry ratio of lung tissue weight (W/D) and the injured alveoli rate (IAR) were measured at 180 minutes after lung reperfusion. Meanwhile the lung tissue slide was prepared for electron microscopic observation at 180 minutes after reperfusion.

RESULTSHO-1 expression was upregulated in two groups in the pulmonary endothelial cells, part of pulmonary vascular smooth muscle cells, extima of vessels and epithelial cells of airway, the absorbance was 0.168 +/- 0.016 (0.148 +/- 0.013), 0.186 +/- 0.014 (0.158 +/- 0.012) respectively.The LGTI group showed higher absorbance than those of the I/R group (P < 0.01), lower W/D and IAR values than those of the I/R group (P < 0.01) significantly and lighter abnormal changes of the lung tissue in morphology than those of the I/R group.

CONCLUSIONLigustrazine injection possesses notable protective effects on I/R in rabbits by increasing the expression of HO-1 in lung.

Animals ; Disease Models, Animal ; Heme Oxygenase-1 ; metabolism ; Lung ; blood supply ; metabolism ; Pyrazines ; pharmacology ; Rabbits ; Reperfusion Injury ; metabolism

AIMTo explore the effect of the ginkgo bioba extract on the expression of heme oxygenase-1 (HO-1) in bronchial asthma.

METHODS30 guinea pigs were randomly divided into 3 groups (n = 10): (1) Normal control group; (2) Asthmatic group; (3) Therapeutic group. Blood carbon monoxide Hb (COHb) percent value, Airway resistance and eosinophilic inflammation of airway wall were observed, the expression of HO-1 in lung tissue were observed by immunohistochemical staining.

RESULTSThe expression of HO-1 was mainly located in airway epithelium in these 3 groups, the optical densities were 0.170 +/- 0.020, 0.707 +/- 0.058, 0.397 +/- 0.034, respectively. The asthmatic group showed higher optical densities than that of the normal control group (P < 0.01), and the therapeutic group showed lower optical density than asthmatic group (P < 0.01).

CONCLUSIONThe expression of HO-1 is inhibited significantly by the treatment of the ginkgo bioba extract, which may be one of the mechanism for treating asthma by ginkgo bioba extract.

Animals ; Asthma ; drug therapy ; metabolism ; Ginkgo biloba ; Guinea Pigs ; Heme Oxygenase-1 ; metabolism ; Phytotherapy ; Plant Extracts ; pharmacology

This project aimed to explore the protective effect of ginsenoside Rg_1 on hypoxia/reoxygenation(H/R)-induced H9 c2 cardiomyocyte injury and its underlying signaling pathway. The H/R model of H9 c2 cardiomyocytes was established and then the cells were divided into different treatment groups. CCK-8(cell counting kit-8) was used to detect the activity of cardiomyocytes; Brdu assay was used to detect the proliferation of H9 c2 cells; the caspase-3 activity was tested, and then the protein expression was assessed by Western blot. Flow cytometry was used to evaluate the apoptosis level of cardiomyocytes. Ginsenoside Rg_1 inhibited H/R-induced cardiomyocyte apoptosis and caspase-3 activity, promoted nuclear transcription of nuclear factor erythroid-2 related factor 2(Nrf2), and enhanced the expression of the downstream heme oxygenase-1(HO-1). Ginsenoside Rg_1 could increase Nrf2 nuclear transcription and HO-1 expression with the increase of concentration(10, 20, 40, 60 μmol·L~(-1)). However, the protective effect of ginsenoside Rg_1 on cardiomyocytes was significantly weakened after the transfection of Nrf2-siRNA. Ginsenoside Rg_1 could protect cardiomyocytes by activating the Nrf2/HO-1 pathway.
Apoptosis ; Ginsenosides/pharmacology* ; Heme Oxygenase-1/genetics* ; Humans ; Hypoxia ; Myocytes, Cardiac ; NF-E2-Related Factor 2/genetics*

Heme oxygenase (HO) is a rate-limiting enzyme for endogenous carbon monoxide (CO) production. Recently it has been suggested that endogenous CO plays an important role in regulating smooth muscle tone. The development of bladder outlet obstruction in men with benign prostates hyperplasia was shown to be related to the prostates smooth muscle tone, but it was not clear whether endogenous HO/CO system mediates prostates smooth muscle activity. To investigate the influence of sex hormone on the expression of heme oxygenase (HO) gene in rat ventral prostate, we created the model of castrated male rats to test the mRNA levels of HO-1 and HO-2 by RT-PCR, and used immunohistochemical staining procedures with image analytic technical system to confirm the effects of exogenous androgen and estrogen on the expression of HO-1 and HO-2 protein in rat ventral prostate. The results showed that two isoforms of HO were present in rat ventral prostate. The epithelial cells of acini and fibromuscular stroma of the rat prostate displayed HO-1 immunoreactivity, whereas HO-2 immunostaining was only examined to be in the acinar cells. Both at protein and transcript levels, HO-1 in castrated group was markedly decreased compared with the normal control group (p<0.01). In groups of exogenous administration of androgen and estrogen HO-1 was much higher than that in the control groups (p<0.01). However, estrogen increased HO-1 protein level in prostate stroma, while the levels of HO-2 did not give any evidence of change among all groups (p>0.05). These findings suggest that expression of HO-1 gene is induced by sex hormones, in contrast, there is no change in HO-2 expression. We speculate that CO-HO system is possibly involved in the pathologic processes of prostates abnormal proliferation induced by sex hormones and that CO derived from HO-1 may play an important role in the regulation of smooth muscle activity in rat prostate.
Animals ; Carbon Monoxide ; metabolism ; Heme Oxygenase (Decyclizing) ; biosynthesis ; genetics ; Heme Oxygenase-1 ; Male ; Orchiectomy ; Prostate ; enzymology ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Testosterone Propionate ; pharmacology

The expression, activity and clinical implication of heme oxygenase-1 (HO-1) in the chronic renal insufficiency (CRI) rat kidney and its mechanism were investigated. The 5/6 nephrectomized rats were assigned to sham operation group, CRI group and Hemin group. At the 8th week after second operation, blood pressure, urinary protein, serum creatinine (Scr) and BUN were measured. Renal pathologic changes were observed. The activity of HO and contents of erythropoietin (EPO) in serum and renal tissue were determined. Immunohistochemistry was used to detect the expression and distribution of HO-1 in the CRI rat kidney. As compared with CRI group, the urinary protein, blood pressure, Scr and BUN in Hemin group were reduced significantly (P < 0.05). The glomerular mesangial proliferation, inflammatory cellular infiltration of renal interstitium and interstitial fibrosis were ameliorated significantly. Immunohistochemistry and measurement of HO-1 activity revealed that the expression and activity of HO-1 was decreased in renal tissues and increased in serum in CRI group as compared with normal rats. HO-1 distributed mainly in tubular epithelial cells. The EPO contents in Hemin group were significantly higher than in CRI group. Through up-regulating the EPO level in serum and renal tissues, HO-1 retards the progression of CRI.
Animals ; Erythropoietin ; metabolism ; Heme Oxygenase (Decyclizing) ; metabolism ; Heme Oxygenase-1 ; Hemin ; pharmacology ; Kidney ; drug effects ; enzymology ; pathology ; Kidney Failure, Chronic ; enzymology ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Up-Regulation

OBJECTIVETo investigate the effects of Salvia miltiorrhiza (SM) extracts on the expression of heme oxygenase-1 (HO-1) and nitric oxide synthase (NOS) in small pulmonary arteries (SPAs) of rats with chronic hypoxia.

METHODSAfter two weeks of hypoxia, rats were treated with diltiazem, and small, median, and large doses of SM extracts. The lungs were tested for the expression and distribution of HO-1, endothelial NOS (eNOS) and inducible NOS (iNOS) by using immunohistochemistry and Western blot method.

RESULTSMedian and large doses of SM extracts significantly reduced hypoxia-induced media thickening in the SPAs (similar with diltiazem), recovered repaired ultrastructure injury, decreased HO-1 and iNOS levels, and increased eNOS expression in the SPAs.

CONCLUSIONSMedian and large doses of SM extracts play significant roles in inhibiting structural remodeling in rats with hypoxic pulmonary hypertension. These effects might attribute to the suppression of HO-1 and iNOS, and the promotion of eNOS expression under the conditions of hypoxia.

Animals ; Drugs, Chinese Herbal ; pharmacology ; Heme Oxygenase (Decyclizing) ; metabolism ; Heme Oxygenase-1 ; Hypertension, Pulmonary ; drug therapy ; enzymology ; Hypoxia ; complications ; Nitric Oxide Synthase ; metabolism ; Rats ; Rats, Wistar ; Salvia miltiorrhiza ; chemistry

This study was aimed to investigate the effect of AMN107 (nilotinib) combined with heme oxygenase-1 (HO-1) inhibitor zinc protoporphyrin IX (ZnPPIX) on chronic myeloid leukemia (CML) cells and its mechanism. Proliferative rate of cells treated with AMN107 (10 µmol/L) and ZnPPIX (10 µmol/L) alone or both for different time was observed by MTT and trypan blue methods; the expression of HO-1 in the control group, ZnPPIX (10 µmol/L) group, AMN107 (10 µmol/L) group, AMN107 (10 µmol/L) combined with ZnPPIX (10 µmol/L) group was evaluated by semi-quantitative RT-PCR and Western blot at 48 h. Cell apoptosis was detected by flow cytometry with Annexin V/PI double staining at 48 h. The results showed that the strongest inhibition of cell proliferation was detected in combined group, and in a time-dependent manner; the expression level of HO-1 was lowest in combined group; the cell apoptosis rates were (11.38 ± 0.02)%, (17.44 ± 0.08)%, (39.81 ± 0.07)% and (56.46 ± 0.19)% in the control group, ZnPPIX group, AMN107 group, AMN107 combined with ZnPPIX group at 48 h respectively. It is concluded that the second-generation tyrosine kinase inhibitor AMN107 can induce the apoptosis in CML cells. Inhibition of HO-1 expression can enhance the killing effect of AMN107 on CML cells, which provides experimental evidence to further improve the clinical efficacy of CML treatment.
Apoptosis ; drug effects ; Heme Oxygenase-1 ; antagonists & inhibitors ; Humans ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; pathology ; Protoporphyrins ; pharmacology ; Pyrimidines ; pharmacology